Our broad objective is to understand the means by which Borrelia burgdorfer (Bb) establishes infection:transmission cycle between the tick vector and mammalian reservoir host, both of which are needed to maintain the spirochete in nature. 1. Outer surface protein function and expression. The outer membrane of Bb contains several abundant lipoproteins (Osps) that vary in size and expression. It is likely that the different Osps confer distinct properties on the spirochete that are pertinent to the different environments in which it must survive. At least three Osps exhibit temperature-related differential expression, as might accompany tick feeding or transmission to a warm-blooded animal. We have developed a mode system for studying regulation of Osp gene expression in E. coli, using osp promoter fusions to the indicator gene lacZ and are screening for trans-acting Bb gene products that alter promoter expression. We have cloned an osp gene family, which contains at least six related members, and are investigating the expression of these genes during the course of an infection. Studies on the biology of Bb and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. We have developed allelic exchange using resistance to the antibiotic coumermycin A1 as the selectable marker. This represents the first demonstration of targeted gene inactivation by homologous recombination in Bb. We are currently using this method to inactivate osp genes in order to study their roles in the infectious cycle of Bb between ticks and mammals. 2. Plasmid structure and replication. We have identified at least five separate 32 kb circular plasmids in Bb, al of which can be maintained within a single bacterium. Each plasmid contains a different allele of a temperature-regulated Osp, and all plasmids contain homologous sequences throughout their lengths. These plasmids may provide a model for the study of plasmid replication and segregation in Bb. We are investigating their potential utility as plasmid vectors. We have identified genes for homologs of the peptide binding component of oligopeptide permease (OppA) on circular and linear Bb plasmids and on the chromosome. In other bacteria, OppA can be a receptor for peptide pheromones that signal adaptive responses to environmental conditions. We are investigating the expression of plasmid and chromosomal oppA genes. We have targeted oppA and additional plasmid genes for gene inactivation by our recently developed system of allelic exchange in Bb.